Regulation of vertebrate globin genes during erythroid differentiation and development is an informative model of higher eukaryotic gene control. An increasingly large body of evidence supports an important role for epigenetic mechanisms in the control of vertebrate globin gene expression. This project is aimed at elucidating the role and mechanism of DMA methylation in the developmental regulation of embryonic and fetal globin gene expression in adult erythroid cells. The long-term goal of this project is to characterize mechanistic components of developmental globin gene regulation in order to identify selective molecular targets for safe therapeutic activation of fetal/embryonic globin gene expression in ?-thalassemia and sickle cell anemia. This objective will be pursued through the following specific aims: 1) To characterize the protein components of erythroid cell methylated cytosine protein binding complex (MeCPC) that binds preferentially to methylated embryonic globin gene DMA sequences and verify the functional importance of key components; and 2) To determine the mechanism(s) through which methylated cytosine-binding domain protein 2 (MBD2) developmentally silences the human ?-globin gene in a transgenic mouse model and test its function in erythroid cells derived from CD34+ human hematopoietic progenitors. The experimental plan to achieve these specific aims will include transgenic mouse model systems containing single-copy yeast artificial chromosome (YAC) and bacterial artificial chromosomes (BAG) harboring the human ?-globin locus; chromatin immunoprecipitation assays; biochemical purification methods; protein identification by SDS gel separation and ion spray mass spectroscopy; and gene knock-down by small inhibitory RNA (SiRNA) in primary human erythroid cells and cultured cell lines. The rationale for selection of transgenic mouse models and primary erythroid cells for most of the proposed assays is to identify mechanisms through which DMA methylation and other epigenetic controls operate in models that closely resemble those in normal erythroid cells in vivo. Sickle cell anemia and ?-thalassemia are among the most common single-gene-mediated diseases in humans and inflict severe suffering and disability. Natural mutations that result in activation of fetal hemoglobin are known to overcome the molecular deficits in ?-thalassemia and sickle cell anemia. Understanding the DMA methylation-mediated mechanisms of fetal/embryonic globin gene silencing could lead to more effective treatment of these diseases and could facilitate treatment strategies for other common diseases, including cancer, in which DNA methylation-mediated gene silencing is believed to play a major role. [unreadable] [unreadable] [unreadable]